| 摘要: | 心律不齊引起的心臟猝死(CSD)是造成臺灣每年150 萬個心血管疾病患者的主要 死因之一，佔所有因心血管問題導致的死亡近50％的比率。尤其近年在年輕成人 族群上，突然發生心臟猝死的病例逐年提高，顯示我們對心律不齊所引發心臟猝 死的起因，目前尚未完全了解。 在之前的研究中，我們利用HEK293 細胞來過度表達與調控心律節奏有關的HCN 離子通道，結合電生理的方法，發現酪胺酸磷酸化能調控HCN 離子通道的活性， 包括改變通道的電流密度與電流動力學。然而酪胺酸磷酸化在整體動物水平上， 是否會調控心律節奏以及引發心動過速或心動過慢所造成的心律不齊，目前不得 而知。再者，由於一直缺乏可靠與可重複性的動物模型來提供我們探討心律不齊 的機制，所以增加了研究心律不齊以及相關新藥篩選的困難度。 第一年的研究我們主要是利用hemodynamic measurements 的技術在大白鼠體內 注入酪胺酸去磷酸酶抑制劑sodium orthovanadate (Na3VO4)以及酪胺酸酶抑制劑 PP2，來增加或降低酪胺酸磷酸化的程度。藉此來a) 研究在整體的動物水平上， 改變酪胺酸磷酸化是否具有調控心律以及心血管的功能，b) 建立一個新的動物 模型來研究心律不齊，以及c) 提供新藥篩選的平台，來降低或延遲心律不齊的 發生率。 第二、三年的研究中，我們將利用生化分析以及共軛焦顯微鏡技術，在心肌細胞 與HEK293 細胞上來探討其背後可能的機制，包括a) 藉由Na3VO4 來提高酪胺 酸磷酸化水平，探討是否影響HCN4 離子通道的整體蛋白水平表現以及其上膜 量， b) 藉由PP2 來減少酪胺酸磷酸化水平，探討是否影響HCN4 離子通道的 整體蛋白水平表現以及其上膜量，c) 藉由heart rate variability 的分析來探討自主 神經系統是否受到Na3VO4/ PP2 改變酪胺酸磷酸化的影響。
Cardiac arrhythmias caused Cardiac sudden death (CSD) was a major factor affects approximately 1,500,000 people of cardiovascular patients in Taiwan, and is estimated to account for nearly 50 percent of all death from cardiovascular causes. However, there are also cases where people, especially young adults, died of CSD in the absence of defective heart structure including hypertrophy. This indicates that our knowledge of CSD is not a complete picture. From a pathological point of view, it is commonly accepted that the underlying cause of CSD is tachycardia-induced arrhythmias. Therefore, to obtain a comprehensive understanding of arrhythmias is the key to understand the causes of CSD. However, the underlying mechanisms of cardiac arrhythmias still remain obscure due to lack of a good animal model system with reliably inducible and controlled real-time cardiac arrhythmias. In previous studies, we employed HEK293 to overexpress the pacemaker Hyperpolarized Cyclic-Nucleotide gated (HCN) channels combined with electrophysiology method (whole cell recording) to study the regulation of HCN channel activities by tyrosine phosphorylation. Our results indicate that either current densities or current kinetics of HCN channels can be modulated by tyrosine phosphorylation. However, if and how cardiac rhythm and/or tachycardia-/bradycardia -induced cardiac arrhythmias can be regulated by tyrosine phosphorylation in whole animal model are still unknown. Furthermore, lacking reliable and reproducible animal models for us to study the underlying mechanisms of cardiac arrhythmias contributes the difficulties of our understanding of CSD and the screening for developing the therapeutic medicines. In first year, we will employ hemodynamic measurements and administrate both tyrosine phosphatase inhibitor, sodium orthovanadate (Na3VO4), and/or tyrosine kinase inhibitor, PP2, to increase and/or decrease endogenous tyrosine phosphorylation in SD rats in order to a) investigate if cardiac rhythm and cardiovascular function (for example, systolic-/diastolic- left ventricular pressure, blood pressure, ±dp/dt, and ECG etc.) can be regulated by tyrosine phosphorylation. b) establish a rat model with controllable occurrence of cardiac arrhythmias without cardiac structural remodeling. c) provide a reliable platform to study the underlying mechanisms of cardiac arrhythmias and to develop novel antiarrhythmic drugs for treatment of patients without structural heart disease. This platform will be useful in effort to test small molecules or nature/chemical compounds to delay and ultimately decrease the incidents of cardiac arrhythmias. In order to further investigate the underlying mechanisms of effects of tyrosine phosphorylation on the regulation of cardiac rhythm, for second and third year, biochemistry approaches (i.e. western blot, co-immunoprecipatation, and biotinylation) and confocal image technology will be used. By employing both cardiac myocytes and HEK293 cells with/without administration of sodium orthovanadate (Na3VO4) and PP2 in comparison with basal, we are going to investigate a) if and how increased tyrosine phosphorylation by sodium orthovanadate (Na3VO4) applying increase the expression of total and membrane channel proteins of HCN4. b) if and how decreased tyrosine phosphorylation via PP2 applying decrease the expression of total and membrane channel proteins of HCN4. c) effects of Na3VO4-/PP2-mediated increased/decreased tyrosine phosphorylation on the activities of autonomic nervous systems via analysis of heart rate variability. |
